Temporal variations in protein tyrosine kinase activity in leukaemic cells: response to all-trans retinoic acid.

Protein tyrosine kinases (PTKs) play a critical role in the modulation of a wide variety of cellular events such as cell division, differentiation and metabolism. Regulation of PTK activity must be tightly controlled as over-stimulation is known to impair normal cell growth, resulting in oncogenic transformation. Since evidence suggests that dynamic oscillatory behaviour occurs in metabolic control processes, we investigated the patterns of oscillatory behaviour in the total protein content and enzyme activity of PTK exhibited by proliferating and differentiating human acute promyelocytic cells. Distinct rhythmic patterns of oscillatory behaviour were observed in both the amount of extractable protein and PTK enzyme activity. Rhythmic characteristics such as period and amplitude were significantly modulated following treatment with all-trans retinoic acid, an inducing agent. These results support the view that dynamic oscillatory control processes may play an important role in regulating cellular behaviour.

Modification of oscillatory behaviour of protein tyrosine kinase and phosphatase during all-trans retinoic acid-induced differentiation of leukaemic cells.

Granulocytic maturation of human acute promyelocytic leukaemic (HL-60) cells was induced using all-trans retinoic acid (ATRA). Time-dependent changes in the enzyme activities of protein tyrosine phosphatase (PTP) and protein tyrosine kinase (PTK), and the total extractable protein content were monitored in proliferating and differentiating cells. The existence of periodicity was demonstrated clearly in both PTP and PTK enzyme activities and in the amount of protein extracted from the cells. Following ATRA treatment, differentiation-induced changes in rhythmic characteristics such as period and amplitude were evident. A noticeable effect was that of ATRA on the enzyme activity of PTP, for which four distinct patterns of oscillatory behaviour were identified. This study examines these changes, in an attempt to gain insight into the role which biochemical oscillators may play in the regulation of molecular control mechanisms.

The influence of lysis buffer composition on the expression and activity of protein tyrosine phosphatase.

Lysis conditions are crucial in the extraction and solubilization of phosphotyrosine-containing proteins in a form that is immunoreactive, undegraded and enzymatically active. To establish optimal experimental conditions, we evaluated protein tyrosine phosphatase enzyme activity and the detection of PTP-1 B protein in human acute promyelocytic leukaemic cells, both before and after retinoic acid treatment. We found that the composition of the lysing buffer greatly influenced the efficiency of solubilization, resulting in major alterations in the activity of protein tyrosine phosphatases and on the mobility of PTP-1 B protein.

Temporal variations in protein tyrosine phosphatase activity during cell proliferation and differentiation.

Cellular oscillations in total extractable protein content and protein tyrosine phosphatase activity were analysed in proliferating and differentiating human acute promyelocytic leukaemic (HL-60) cell extracts. Differentiation was induced along the granulocytic pathway using all-trans retinoic acid (ATRA). High frequency rhythms with distinct, well defined waveforms of varying amplitude were observed for both the total protein content and for the enzyme activity of protein tyrosine phosphatase (PTP). Linear correlation analysis showed that there was no obvious relationship between the protein content and the corresponding PTP activity, suggesting that the periodic variations between these two components are relatively independent of each other. ATRA significantly altered the characteristics of the rhythms with respect to the period and amplitude and had a dampening effect on the oscillatory pattern of PTP activity. Modulation of such characteristics may be of significance with respect to the regulation of the differentiation processes and the possible reversal of transformation.

Evidence for a phytoreovirus associated with tobacco exhibiting leaf curl symptoms in South Africa.

ABSTRACT Three forms of tobacco leaf curl (termed classes I, II, and III, based on symptomatology) recently have been described in southern Africa. Numerous attempts to isolate virus particles responsible for a nongeminivirus-induced leaf curl disease (class I) of tobacco in South Africa have been unsuccessful. Recently, 12 dsRNA segments were isolated from tobacco exhibiting class I leaf curl symptoms, suggesting a possible reovirus genome. The objective of our study was to confirm whether the dsRNA segments are associated with a reovirus. Isolation of icosahedral particles with an outer core 60 to 65 nm in diameter and an inner core 40 to 45 nm in diameter was achieved. Twelve distinct nonpolyadenylated dsRNAs were isolated from purified virions, and the total molecular masses of the dsRNAs ranged from 17.86 to 18.40 x 10(6) Da in polyacrylamide and agarose gels, respectively. Using hybridization analysis, dsRNAs were identified as non-homologous distinct segments. Comparisons with other known reoviruses revealed a unique banding pattern that was most similar to the wound tumor virus (WTV), the type species of the genus Phytoreovirus. Hybridizations of WTV cloned DNA probes (segments S4 and S6 to S9) and dsRNAs from infected tobacco indicated no significant sequence similarity, whereas indirect enzyme-linked immunosorbent assay with a polyclonal antiserum to WTV showed strong positive cross-reactivity to tobacco virions. Our results indicate a virus with features consistent with those of phytoreoviruses. This is the first report of a plant reovirus in tobacco, the first record in Africa, and the second example of a field-isolated dicot phytoreovirus.